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Vortex AMPure XP beads frequently to make sure that beads are evenly distributed.Īdd 50 μl of AMPure XP beads to each well. In a 96-well plate, pipette 45uL of prepared master mix per sample.Īdd 5uL of extracted DNA template (~150 ng template ( 16SFULL (bold indicates 30 cycles) If your primers are not this concentration, adjust the volume added. Our primers are at this concentration in the freezer. Note: The original IDT stock solutions of the primers are at a concentration of 100 uM. Prepare the following, per sample, as a master mix:.Step 2: PCR Amplify the full-length 16S rRNA gene Extract DNA using QIAGEN DNeasy kit (bacterial protocol) or other method of bacterial DNA extraction.Ampure XP Beads (remove from fridge and acclimate at room temp for 30 min before use).Molecular biology grade, nuclease-free water (-20C or room temperature).Combining the seqeunce data for the three amplicons generates a contiguous sequence that spans that majority of the full 16S rRNA gene, therby giving you a decent idea of bacterial identity. The protocol uses three primers (two forward and one reverse) to generate amplicons for Sanger sequencing.

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